Production of transgenic mice and rabbits that carry and express the human tissue plasminogen activator cDNA under the control of a bovine alpha S1 casein promoter

One-cell embryos from mice and rabbits were microinjected with a hybrid gene composed of 1.6 kilobases (kb) promoter/regulatory sequences of the bovine alphaS1 casein gene fused to the complementary DNA (cDNA) encoding for the human tissue plasminogen activator (htPA) and 3'untranslated sequences from rabbit beta-globin and SV 40 genes. Transgenic mice and rabbits that carry the htPA gene were obtained. In mice, 11 founder females were generated, and 6 of them expressed low levels (about 50 mug/ml) of htPA in their milk. Some of the transgenic mice showed rearrangements of the microinjected DNA sequences as judged by Southern blot analysis. A position-dependent expression of the transgene is suspected to occur. The only live-born founder transgenic rabbit obtained was a male, and it transmitted the transgene in a Mendelian fashion to F1 females, which expressed htPA at very low levels (8 to 50 ng/ml). Although the 1.6-kb bovine alphaS1 casein promoter that was used directs the synthesis of htPA specifically to the mammary gland, it may not be sufficient for a high level of expression.     

Increased Milk Yield in Transgenic Mice Expressing Insulin-like Growth Factor 1

Abstract

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine α-lactalbumin (α-LA) promoter linked to an ovine IGF-1 cNDA. This α-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine β-casein gene in the lactating mouse mammary gland

The spatial, temporal, and hormonal pattern of expression of the β-casein gene is highly regulated and confined to the epithelial cells of the lactating mammary gland. Previous studies have shown that 1.7 kb of the bovine β-casein promoter were able to drive cell-specific and hormone-dependent expression to a mouse mammary cell line but failed to induce accurate expression to the mammary gland of transgenic mice. We investigated here the ability of 3.8 kb of the bovine β-casein gene promoter to drive the expression of the human growth hormone (hGH) gene in transgenic mice. A Northern blot analysis using total RNA obtained from different tissues of lactating and nonlactating females revealed the presence of hGH mRNA only in the mammary gland of lactating females. hGH mRNA was not detectable in the mammary gland of virgin females or males. A developmental analysis showed that hGH mRNA only peaked on parturition, resembling more closely the bovine β-casein temporal expression pattern rather than the murine. In situ hibridization studies performed on mammary gland sections showed that the cellular pattern of hGH expression was homogeneous in all lobules from heterozygous and homozygous transgenic mice. Silver grain counts on the tissue sections highly correlated with the hGH contents in the milk determined by radioimmunoassay (r = 0.996). Thus 3.8 kb of the bovine β-casein promoter direct a high-level expression of a reporter gene to the lactating mammary gland of transgenic mice in a tissue-specific and developmentally regulated manner. Mol. Reprod. Dev. 49:236–245, 1998. © 1998 Wiley-Liss, Inc.     

The High-Level Expression of Human Tissue Plasminogen Activator in the Milk of Transgenic Mice with Hybrid Gene Locus Strategy

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.     

Establishment and initial characterization of the ovine mammary epithelial cell line nish

Summary Analysis of the molecular mechanisms involved in the differentiation and formation of the characteristic three-dimensional structures of the developing mammary gland of the major milk-producing livestock (ducts, end buds, and alveoli) requires in vitro model cell cultures. The few cell lines that have been established from dairy animals do not fully reproduce the entire program of mammary differentiation. Here we present the initial characterization of a unique mammary epithelial cell line derived spontaneously from midpregnant sheep (NISH). These cells form in vitro functional structures resembling ducts, lateral buds, and alveoli that secrete β-lactoglobulin (BLG) in an ECM (extracellular matrix)-dependent manner. Interestingly, the presence of growth hormone dramatically increased BLG secretion from NISH cells cultured on ECM. It appears that GH is required not only to establish the structural organization but also is continuously needed to maintain BLG expression. Stable transfection of NISH cells with BLG/Human Serum Albumin (HSA) hybrid gene constructs revealed that the relative level of expression was comparable to the in vivo secretion of HSA in transgenic mice carrying these gene sequences. No expression could be detected in cells transfected with hybrid genes carrying either HSA cDNA or the entire HSA gene, and HSA expression was dependent on the presence of intronic sequences. These results demonstrate that NISH cells may prove a useful tool for studying the differentiation and organogenesis of mammary epithelial cells under defined culture conditions. Furthermore, transfected NISH cells may be an alternative for the transgenic mouse model in evaluating the potential of gene constructs to be efficiently expressed in the mammary gland of transgenic farm animals.     

转溶菌酶基因山羊乳腺上皮细胞的原代培养及特性分析

采用组织块法、胰酶差速消化法分离了人溶菌酶(hLYZ)转基因山羊的乳腺上皮细胞,建立了转基因羊乳腺上皮细胞(TGMEC)的体外培养体系,并对其生长与体外分泌特性进行了分析.研究显示其体外生长曲线符合典型的S型,传代期间二倍体染色体数正常;通过免疫荧光染色、RT-PCR发现该细胞能表达上皮细胞所特有的角蛋白18,并能表达内源性乳蛋白(酪蛋白与乳球蛋白);进一步通过酶联免疫试验发现该乳腺上皮细胞在体外培养中可持续稳定地分泌重组人溶菌酶.由此获得的人溶菌酶转基因羊乳腺上皮细胞系为乳腺生物反应器研究提供了一个重要细胞膜型.     

Expression and characterization of functional recombinant bovine follicle-stimulating hormone (boFSHalpha/beta) produced in the milk of transgenic rabbits

Bovine follicle-stimulating hormone (boFSH) is a heterodimeric glycoprotein that belongs to the pituitary gonadotropins. Bioactive FSH is composed of alpha and beta subunits which require extensive N-glycosylation and sialylation. The mammary gland of transgenic livestock is an attractive source for the synthesis of post-translationally modified proteins. Two mammary gland-specific gene constructs with the cDNA for the boFSH alpha (boFSHalpha) and beta (boFSHbeta) subunits controlled by bovine alpha-s1 casein regulatory sequences were co-microinjected into fertilized rabbit oocytes. Two FSHalpha/FSHbeta double transgenic rabbit lines were established. The transgene expression was strictly lactation and mammary gland specific. Protein analysis revealed the presence of the boFSH heterodimer in the milk of transgenic rabbits showing a molecular weight similar to that of purified pituitary gland derived boFSH (boFSH-P). Subunit specific antibodies detected both polypeptides with the expected molecular sizes. Biochemical characterization demonstrated the expected isoelectric points of the recombinant boFSH. The presence of the post-translationally added terminal sialic acid residues was indicated by wheat germ agglutinin (WGA) lectin Western blotting. The biological activity of the recombinant mammary gland produced boFSH was determined using a FSH-dependent reporter cell line. The bioactivity of the recombinant boFSH was comparable to that of purified boFSH-P.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications. The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors. We established an efficient gene transfer and nuclear transfer approach in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer and some of reconstructed embryos could develop into blastocyst in vitro. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1513–1519 [译自: 遗传]     

Expression of beta‐galactosidase and pig leptin gene in vitro by recombinant adenovirus

Abstract

Adenovirus has been used in vivo and in vitro as a vector to carry a foreign gene for gene transfer. Two kinds of replication defective human recombinant adenovirus vectors were used in this study, the first containing β‐galactosidase reporter gene (AdCMVLac‐Z) and the second carrying a gene for porcine leptin gene (AdCMVpLeptin). AdCMVLac‐Z was tested for its ability to transfer DNA into pig kidney and pituitary cells. These cells expressed Lac‐Z transiently 48 hours after the infection. In addition, when the pig kidney cells expressing the Lac‐Z were replated with low density for the formation of colonies from each cell, colonies of blue cells expressing Lac‐Z were observed. These results demonstrate that human recombinant adenovirus can be used as a transducing viral vector for inducing long‐term expression in pig kidney cells. We also constructed a recombinant adenovirus (AdCMVpLeptin) which contained a pig leptin gene for the expression of pig leptin in vitro in the 293 human kidney cell line. 293 cells transfected with AdCMVpLeptin produced both a 15 KDa of a secretory form of porcine leptin and an 18 KDa long form containing signal peptide. Our study demonstrated that the recombinant adenovirus system offers a method for gene transfer and expression in pig cells.     

山羊β-casein位点打靶载体在乳腺上皮细胞中的表达研究

以本地山羊基因组DNA为模板,通过长链PCR扩增出山羊β-casein上游包括启动子,外显子1及部分外显子2的6.1kb的调控序列及下游3.3kb的序列,将来自质粒pCDNA3的neo基因以及来自质粒pNEOZTK-2的tk基因,经克隆重组后构建了本地山羊乳腺特异性定点打靶载体,并在其中克隆人乳铁蛋白mini基因,采用脂质体法转染小鼠乳腺上皮癌化细胞系C127,以进行打靶载体的表达功能检测,双夹心ELISA测得诱导液中乳铁蛋白表达量为0.2μg/mL,Western-blot显示重组蛋白分子量比标准品略小,约为76kD,结果说明本载体能够指导外源基因在动物乳腺细胞内正确表达。     

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

A Chinese Hamster Ovary cell line, CHO1-15₅₀₀, producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell⁻¹ h⁻¹) and early decline (0.040 pg cell⁻¹ h⁻¹) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h⁻¹. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.     

RT-PCR测定组织纤溶酶原激活剂突变体(La-tPA)在转基因鼠乳腺表达的研究

利用所构建的乳腺表达组织纤溶酶原激活剂突变体（Ｌａ－ｔＰＡ）载体．对５４０枚小鼠受精卵进行显微注射,经ＰＣＰ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ转基因小鼠．为了精确研究转基因在小鼠体内的表达,采用ＲＴ－ＰＣＰ方法测定转基因鼠在泌乳期１～２０ｄＬａ－ｔＰＡ基因的转录,结果表明,在转基因鼠乳腺的Ｌａ－ｔＰＡｍＲＮＡ水平在１０～１５ｄ最高,在２０ｄ时降为最低．外源基因在转基因小鼠乳腺的表达规律研究为未来利用转基因动物生产Ｌａ－ｔＰＡ提供依据     

High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2–630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.     

tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

人组织纤溶酶原激活剂(tissue-type plasminogen activator,tPA)是一种被广泛应用于临床的溶栓药物。双基因共整合入生物体内能够产生协同作用,从而提高目的基因的表达水平。但是目前,利用gGH基因与tPA基因共整合以期提高tPA表达水平的相关研究较少。为筛选获得tPA高表达的tPA/gGH双基因整合的单克隆转基因山羊乳腺上皮细胞株,本研究以β-casein基因作为调控序列,构建乳腺特异性表达载体PCL25/gGH,并通过电转染将tPA和gGH双基因共转染山羊乳腺上皮细胞;通过G418筛选获得抗性细胞株,经PCR检测获得转基因单克隆细胞株;利用催乳素诱导tPA表达,收集48 h后细胞诱导液进行ELISA()和Western blotting检测并分析其tPA表达水平。结果表明,共获得142株抗性单克隆细胞,其中有53株tPA单基因整合细胞株,34株tPA/gGH双基因整合细胞株,双基因整合率达23.9%(34/142)。共检测出29株细胞能够表达tPA,其中单基因表达细胞为12株,表达率为22.6%(12/53);双基因表达细胞为17株,表达率为50.0%(17/34);且单基因细胞表达tPA含量为7.5~52.0μg/mL,而双基因细胞表达tPA含量为40~360μg/mL,明显高于单基因表达水平。本研究通过电转染的方式成功获得了tPA/gGH双基因整合的单克隆山羊乳腺上皮细胞株,并证明双基因整合的细胞株表达tPA水平明显提高,为后期制备高表达转基因山羊奠定了基础。     

Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant chinese hamster ovary cells

Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture. (c) 1994 John Wiley & Sons, Inc.     

Dynamic Control of Oligosaccharide Modification in the Mammary Gland: Linking Recombinant Human Erythropoietin

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin α and β are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and α1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin α derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin α. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use. These authors are equal contributors to this work.     

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.     

Expression of a Truncated Brca1 Protein Delays Lactational Mammary Development in Transgenic Mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.